Microsatellite Markers for Two Species of Dampwood Termites in the Genus Zootermopsis (Isoptera: Termopsidae)

Molecular Ecology Notes (2004) doi: 10.1111/j.1471-8286.2004.00791.x

PRIMERBlackwell Publishing, Ltd. NOTE Microsatellite markers for two species of dampwood termites in the genus Zootermopsis (Isoptera: Termopsidae)

BENJAMIN T. ALDRICH and SRINIVAS KAMBHAMPATI Department of Entomology, Kansas State University, Manhattan, KS 66506 USA

Abstract Dampwood termites in the genus Zootermopsis inhabit forested areas in western North America. To better understand the colony composition and breeding structure of Zootermopsis, we identified polymorphic microsatellite loci to use in population analysis. Microsatellite loci were isolated from Zootermopsis nevadensis nevadensis (Hagen); however, all primers amplified homologous loci in Zootermopsis angusticollis (Hagen) and Zootermopsis nevadensis nuttingi (Hagen). Twelve loci were polymorphic in one or more of the above subspecies and species. The number of alleles per locus ranged from one to six, with some allelic differences among subspecies and species. We are currently utilizing the microsatellite markers to investigate the population genetics of Zootermopsis. Keywords: breeding structure, gene flow, microsatellite, Zootermopsis Received 2 June 2004; revision received 3 August 2004; accepted 3 August 2004

Dampwood termites of the genus Zootermopsis occur in the brief, four µg of genomic DNA was digested with RsaI and forests of the Cascade and Sierra Nevada mountains in the HaeIII to generate approximately 500 bp fragments and western United States. Three species are known to occur in ligated to Snx linker primers (SnxF: 5′-CTAAGGCCTT- this area: Z. angusticollis (Hagen), Z. nevadensis (Hagen), and GCTAGCA GAAGC-3′ and SnxR: 5′-GCTTCTGCTAG- Z. laticeps (Banks). Zootermopsis nevadensis is subdivided CAAGGCCTTAGAAAA-3′). Digested DNA with linkers into two subspecies based on cuticular hydrocarbon pheno- was then hybridized to microsatellite oligos (probes) types: Z. nevadensis nevadensis and Z. nevadensis nuttingi and removed from supernatant using Avidin D beads (Haverty & Thorne 1989). Although much is known about and affinity chromatography. Bound DNA was eluted and the biology of Zootermopsis (reviewed by Weesner 1969), polymerase chain reaction (PCR)-amplified using the Snx little information is available on their colony composition, linker primers. Using a TA Cloning Kit (Invitrogen Inc.), population genetics, and breeding structure. It is not known enriched DNA fragments were cloned following the man- whether the two subspecies interbreed to form hybrids and ufacturer’s directions. Positive clones were identified and if the species or subspecies vary in their overall breeding the inserts were PCR-amplified with M13 primers (M13F: structure. Furthermore, the number of reproductives 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′ and M13R: 5′- per colony and relatedness among colony members has TCACACAGGA AACAGCTATGAC-3′). PCR was set up µ µ µ not been estimated. To compare the breeding structure in 31 L volumes containing: 22.5 L H2O, 2.5 L of 10X of Zootermopsis species, estimate levels of gene flow, and Taq buffer (500 mm KCl, 100 mm Tris-HCl pH 9.2, 1% µ µ determine the number of reproductives per colony, we Triton X-100), 2.5 L MgCl2 (2.5 mm), 0.25 L dNTPs (80 mm) developed primers for 12 polymorphic microsatellite loci. (Promega), 0.4 µL of each primer (0.25 µg/µL), 0.4 µL of Taq DNA was extracted from the head and thorax of Z. n. DNA polymerase (Promega), and 2 µL of DNA. Amplifi- nevadensis collected in northern California. Microsatellite- cation was achieved using a PTC-200 thermal cycler (MJ enriched DNA was prepared using the approach developed Research) with the following conditions: an initial denatura- in the laboratory of Travis Glenn (http://www.uga.edu/ tion step of 94 °C (3 min) followed by 35 cycles of 95 °C ∼srel/Msat_Devmt/Microsatellite_Development.htm). In (30 s), 52 °C (45 s), and 72 °C (45 s) followed by 72 °C for 10 min. The PCR product was visualized on an 1% agarose Correspondence: Benjamin T. Aldrich. Fax: (785) 532 6232; E-mail: gel to determine if an insert was amplified. Fragments con- [email protected] taining inserts greater than 400 bp were purified using the

2 PRIMER NOTE

Table 1 Properties of microsatellite loci from Zootermopsis

Locus Forward primer Reverse primer Frag size (bp) Repeat motif TA

Zoot11 CCT GAA GTG ATG GTT AGT CAG TC ATG AAT GAA GCT GGG TCG GA 243 12(GA)593 Zoot18 CTG TCC CTA TGA CAC GAT CAC CGA TAT ACA AGC TTT CCG GAC 230 15(TG)532 Zoot25 CAT TGG ATC CTG GGA ATC AAA CC CCC AGA GTT ACC TAT TCG CTG 339 11(AACCT)583 Zoot28 GGT ATA TTG TAA GGT CAT TTA ACG CTA CAC TTG ACA ATA CAT GAA TTC 191 10(GAT)595 Zoot29 GGA TCA ACG CAA GTG CGT TTT G GGA GCA AGA CAT TCG TAT TTA GGT T 200 7(TGA)534 Zoot31 TGA CGA AAG CAC ACA CTA AAA CAT CTG TCA TTA AAC AAG GAA TGC C 291 6(CAAA)594 Zoot73 CAT AGA AGC CGG AAC AGG GAA CCG AAA GAA GCA CGA TC 232 11(GAA)595 Zoot76 CAG CCT GTT ACT CAC ACA TCC CCC GTT TGT ACC TAC CTC ATT C 164 5(GATT)593 Zoot101 GAA GTG TTG TGG GTT TGG CA TAG CGT TTG ATT CTT CCG CC 190 6(CTGT)595 Zoot103 GCT TCC CTT TCT GAA TCC TGA TCT CTT CAA CTC ACA AGC GC 271 6(TCA)593 Zoot117 CGT CGA TGT TAT GGA GTT CCA G CTC CTA TCT CAT CCC ACA TTA CG 152 6(TAA), 3(GA)596 Zoot212 CGG AAC AAA AGT GAC TAT GAT GG CAA GAT GAT AA CTC TGT GTT CTC 222 7(CA)582

Frag. size: Size (in base pairs) of the original allele obtained from DNA enrichment and sequencing. Repeat motif: The number of repeats indicated is for the original allele obtained from DNA enrichment and sequencing. T: Optimal annealing temperature in °C. A: Observed number of alleles per locus.

QIAquick Gel Extraction Kit (QIAGEN Inc.). The purified for polymorphism (95% criterion; Hartl & Clark 1997). fragments and M13 forward primer were used in sequen- Forty individuals were analysed per colony. PCR condi- cing reactions using the dRhodamine dye terminator tions for testing of polymorphism were identical to the kit (ABI, Inc.) following the manufacturer’s directions. gradient reactions mentioned above; however, the optimal The resulting DNA sample was liapholized and sent to annealing temperature was used instead of the gradient the University of Florida’s DNA Sequencing Laboratory PCR. The primers developed were also tested on Z. angus- for automated sequencing on an ABI 377 sequencer. The ticollis and Z. n. nuttingi to determine if cross-amplification sequences were examined using ABI’s sequence navi- was possible. gator software. Primer regions were selected manually Of the 23 primers pairs tested, 12 displayed scorable flanking the microsatellite repeat and analysed using Oligo- intraspecific variation among Z. n. nevadensis subpopulations Analyser 3.0 (Integrated DNA Technologies, Inc.). Sequences (Table 1). Polymorphic loci were identified for di-, tri-, tetra-, were deposited in GenBank under accession numbers and pentanucleotide repeats. The lowest number of repeats (AY701764–AY701775). for the polymorphic loci was five and the highest 15. The Primers were designed for 34 microsatellite loci. A two- primers also amplified the target loci in Z. n. nuttingi and step PCR (see below) was performed to determine if the Z. angusticollis. All 12 polymorphic loci for Z. n. nevadensis primer pairs amplified the target fragment followed by were also polymorphic in Z. n. nuttingi and 10 of the gradient PCR to determine the optimal annealing tem- 12 were polymorphic in Z. angusticollis. Loci Zoot11 and perature for the various primers. PCR was set up in 32 µL Zoot103 were the only ones that failed to show intraspecific µ µ volumes containing: 22.5 L H2O, 2.5 L of 10X Taq buffer, variability for Z. angusticollis. If the same cross amplification µ µ 2.5 L MgCl2 (2.5 mm), 0.25 L dNTP’s (80 mm) (Promega), occurs for Z. laticeps, primers developed in this study should 0.35 µL of each primer (0.25 µg/µL), 0.5 µL of Taq DNA be useful in studying and comparing the colony composi- polymerase, and 3 µL of DNA. The two-step program con- tion and breeding structure in all Zootermopsis species. sisted of an initial denaturation step of 94 °C (3 min) fol- Table 2 summarizes preliminary analysis on four Z. n. lowed by 10 cycles of 95 °C (30 s); 45 °C (45 s); and 72 °C nuttingi colonies using fstat version 2.9.3 (Goudet 2001). (30 s); then 25 cycles of 95 °C (30 s); 50 °C (45 s); and 72 °C Forty individuals were analysed per colony. Exact tests followed by 72 °C for 20 min. Gradient PCR consisted of an were used to analyse deviations from Hardy–Weinberg initial denaturation step of 95 °C (3 min) followed by 34 equilibrium and linkage disequilibrium using genetic data cycles of 94 °C (30 s), annealing temperatures of 47; 48; 49; analysis version 1.1 (Lewis & Zaykin 2000) as described 50.5; 52.5; 54.5; 56.5; 58; 59; and 60 °C (45 s); and 72 °C (30 s) by Vargo (2003). Observed heterozygosities were not sig- followed by 72 °C for 20 min. nificantly different from heterozygosities expected under Twenty-three of the 32 primer pairs amplified the target Hardy–Weinberg equilibrium for any locus and no linkage fragment. These primers were used in PCR on five Z. n. disequilibrium was observed for any locus pairs. The F- nevadensis colonies collected from Black Mountain, George- statistics show strong substructuring among colonies. town, and El Dorado National Forest, California, to test The number of alleles per colony were consistent with

Table 2 Heterozygosity estimates and F-statistics for four colon- individuals per colony to obtain more precise estimates of ies of Zootermopsis nevadensis nuttingi the breeding structure of Zootermopsis.

Locus HO HE FIS FST FIT Acknowledgements Zoot 29 0.32 0.29 −0.22 0.09 −0.11 Zoot 18 0.01 0.03 0.73 0.03 0.75 We thank Drs Barbra L. Thorne and Michael I. Haverty for provid- Zoot 25 0.43 0.49 −0.27 0.30 0.12 ing Zootermopsis samples. This study was funded by a National Zoot 212 0.00 0.00 0.00 0.00 0.00 Science Foundation grant 9980253 to S.K. This is journal article Zoot 117 0.43 0.68 0.14 0.26 0.37 number 05–44-J of the Kansas Agricultural Experiment Station. Zoot 11 0.51 0.61 −0.21 0.31 0.16 Zoot 73 0.71 0.72 −0.16 0.15 0.02 References Zoot 76 0.29 0.39 0.05 0.20 0.25 − Zoot 101 0.43 0.48 0.22 0.26 0.10 Goudet J (2001) FSTAT, a Program to Estimate and Test Gene Diversities Zoot 103 0.17 0.32 0.12 0.38 0.46 and Fixation Indices Version 2.9.3. Available from http:// Zoot 28 0.68 0.66 −0.16 0.11 −0.02 www.unil.ch/izea/softwares/fstat.html. Zoot 31 0.51 0.56 −0.19 0.23 0.07 Hartl DL, Clark AG (1997) Principles of Population Genetics. Sinauer Mean for all loci 0.37 0.43 −0.12 0.23 0.13 Associates, Inc, Sunderland, MA. Haverty MI, Thorne BL (1989) Agonistic behavior correlated with HO is the observed heterozygosity. HE is the expected hydrocarbon phenotypes in dampwood termites, Zootermopsis heterozygosity. FIS is the mean reduction in heterozygosity of (Isoptera: Termopsidae). Journal of Insect Behavior, 2, 523–543. individuals in subpopulations. FST is the mean reduction in Lewis PO, Zaykin D (2000) GENETIC DATA ANALYSIS: Computer Program heterozygosity of subpopulations in the total population. FIT is the for the Analysis of Allelic Data, Version 1.1. Available from. http:// reduction in heterozygosity of individuals in the total population. lewis.eeb.uconn.edu/lewishome/.

Vargo EL (2003) Hierarchical analysis of colony and population genetic structure of the eastern subterranean termite, Reticulitermes flavipes, using two classes of molecular markers. Evolution, 57, colonies being founded by a single set of parents; however, 2805–2818. the number of genotypes suggested secondary reproduc- Weesner FM (1969) Termites of the Nearctic Region, In: Biology of tives may be present within some colonies. Studies are Termites (eds Krishna K and Weesner FM), Vol. II. pp. 477–525. presently underway using larger number of colonies and Academic Press, New York, NY.